Bottom Line:
T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined.To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection.Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species.

ABSTRACTT cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

Mentions:
By immunological screening of a library of pedigrees segregating thousands of ENU-induced nucleotide substitutions10, we identified one, 5AT161, with an abnormal response to immunization, anti-nuclear autoantibodies, low anti-CD3ε+CD28-induced proliferation and a decreased percentage of naive CD44low CD4+ T cells in the blood (Supplementary Fig. 1a and data not shown). The latter two phenotypes proved to be inherited as a fully penetrant, recessive trait that was readily scored and fixed in subsequent generations. In homozygous 5AT161 mutants, flow cytometry of blood and lymph nodes (LN) showed a reversal of the normal CD4:CD8 ratio and an 8-fold decrease in the absolute number of naïve CD4+ cells and a 3-fold decrease in the naïve CD8+ cells (Fig. 1a). By contrast, the number of antigen-experienced CD44hi cells in both subsets was normal (Fig. 1a). Peripheral naïve CD44lo cells sorted from 5AT161 mice proliferated and upregulated CD25 equivalently to control counterparts following stimulation with anti-CD3ε and anti-CD28 (Supplementary Fig. 1b), confirming that the decreased proliferation in the original screen was due to decreased cellularity. Absolute numbers of other non-T cell hematopoietic lineages (B cells, natural killer (NK) cells, dendritic cells (DCs), and granulocytes) were similar in the central and peripheral lymphoid organs of 5AT161 and control mice, indicating a selective defect in T cell development (Supplementary Fig. 2 and data not shown).

Mentions:
By immunological screening of a library of pedigrees segregating thousands of ENU-induced nucleotide substitutions10, we identified one, 5AT161, with an abnormal response to immunization, anti-nuclear autoantibodies, low anti-CD3ε+CD28-induced proliferation and a decreased percentage of naive CD44low CD4+ T cells in the blood (Supplementary Fig. 1a and data not shown). The latter two phenotypes proved to be inherited as a fully penetrant, recessive trait that was readily scored and fixed in subsequent generations. In homozygous 5AT161 mutants, flow cytometry of blood and lymph nodes (LN) showed a reversal of the normal CD4:CD8 ratio and an 8-fold decrease in the absolute number of naïve CD4+ cells and a 3-fold decrease in the naïve CD8+ cells (Fig. 1a). By contrast, the number of antigen-experienced CD44hi cells in both subsets was normal (Fig. 1a). Peripheral naïve CD44lo cells sorted from 5AT161 mice proliferated and upregulated CD25 equivalently to control counterparts following stimulation with anti-CD3ε and anti-CD28 (Supplementary Fig. 1b), confirming that the decreased proliferation in the original screen was due to decreased cellularity. Absolute numbers of other non-T cell hematopoietic lineages (B cells, natural killer (NK) cells, dendritic cells (DCs), and granulocytes) were similar in the central and peripheral lymphoid organs of 5AT161 and control mice, indicating a selective defect in T cell development (Supplementary Fig. 2 and data not shown).

Bottom Line:
T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined.To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection.Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species.

ABSTRACTT cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.